Approximately 24 million Americans wear soft contact lenses, and clinical studies have demonstrated that overnight use (extended wear) of these lenses is a significant risk factor for corneal infection, which can result in permanent vision loss (Buehler, P., et al., Arch Ophthalmol. 110:1555-1558, 1992; Wilhelmus, K., CLAO J 13:211-214, 1987). The defenses that normally protect the eye against infection are not well understood.
The most common pathogen involved in contact lens-associated corneal infection is Pseudomonas aeruginosa. To cause corneal infection in vivo, P. aeruginosa must bind to the epithelial cell surface; however, before binding can occur the pathogen must resist nonspecific ocular immune mechanisms that help to prevent infection under normal conditions. These include the presence of components with antibacterial properties in the tear film (e.g., lysozyme, lactoferrin, peroxidase, ceruloplasmin, prealbumin, sialin, β-lysin, and cytokines), as well as the sheering force of the blink which wipes the ocular surface, enhances the flow of tears, and facilitates removal of unwanted pathogens from the eye. Furthermore, the innermost layer of the human tear film contains mucus, a glycoprotein gel that serves to improve the spread and stability of aqueous tears, lubricate and protect the underlying corneal epithelium (Holly, F., and Lemp, M., Exp Eye Res 11:239-250, 1971). Research has shown that antibacterial tear film components are not fully effective against P. aeruginosa, however it has been demonstrated that in the open blinking eye, mucins can bind P. aeruginosa and may thereby facilitate bacterial clearance from the ocular surface via normal tear exchange (Fleiszig, S., et al., Infect. Immun. 62:1799-1804, 1994). This mechanical clearance mechanism, however, does not explain why the ocular surface is not normally heavily colonized by bacteria as is the case for other mucous membranes.
After contact with microorganisms, animal studies have shown that Xenopus skin (Zasloff, M., Proc. Natl. Acad. Sci. 84:5449-5453, 1987), and bovine trachea and tongue epithelia (Schonwetter, B., et al., Science 267:1645-1648, 1995) are rich in sources of peptide antibiotics (Boman, H., Annu. Rev. Immunol. 13:61-92, 1995), which could be involved in the unexpected resistance of these tissues to infection. Broad spectrum antimicrobial substances have been demonstrated at several body surfaces that act rapidly to neutralize a broad range of potential pathogenic microbes. One type of antimicrobial substances are the defensins. In general, defensins are characterized as cationic antimicrobial peptides that are generally less than 50 amino acids long, and contain three pairs of disulfide linked cysteines. Three types of defensin polypeptides are known: the classical defensins, the β-defensins, and the insect defensins (see Martin, E., et al., “Defensins and other endogenous peptide antibiotics of vertebrates,” J. Leukocyte Biol. 58:128-133, 1995; Ganz, T., and Lehrer, R. I., “Defensins,” Curr. Opinion Immunol. 6:584-589, 1994, both incorporated herein by reference).
Classical defensins form a triple-stranded β-sheet structure connected by a loop with a β-hairpin hydrophobic finger, and are expressed in granules of myeloid cells such as neutrophils. The β-defensins are cationic peptides with amphiphilic anti-parallel beta sheet structures. The β-defensins differ from classical defensins in the location and connectivity of their conserved cysteines and other conserved amino acids, such as glycine and proline. A human β-defensin (hBD-1) was found to be produced by the urogenital tract epithelia and, to a lesser extent, trachea and lung epithelia (Zhoa, C., et al, FEBS Lett. 396:319-322, 1996). The expression of human β-defensin-1 does not appear to be regulated by the presence of bacteria or inflammatory stimuli (Bensch, K., et al., FEBS Lett. 368:331-335, 1995; Zhoa 1996, supra).
More recently, Harder, J., et al (Nature 387:861, 1997, incorporated herein by reference) showed that human skin expresses an inducible, transcriptionally regulated, antibiotic peptide, which shows homology to bovine tongue- and trachea-derived antimicrobial peptides (Schonwetter et al. 1995, supra). This peptide is characterized by an amphiphilic beta sheet that is believed to bind strongly to target bacteria, insert into the microbial lipid membrane, and thereby alter membrane permeability and internal homeostasis. The peptide, named human β-defensin-2, is the second human β-defensin to be described. Unlike hBD-1, human β-defensin-2 (hBD-2) is regulated at a transcriptional level in response to contact with microorganisms or TNFα, and is effective in killing the gram-negative bacteria P. aeruginosa and Escherichia coli, and the yeast Candida albicans. 